Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Loading in …3
×
1 of 37

Ubiquitin mediated proteolysis

24

Share

Download to read offline

Ubiquitin mediated proteolysis

Ubiquitin mediated proteolysis

  1. 1. ubiquitin PDB 1TBE
  2. 2. • What is ubiquitin mediated proteolysis? • Who discovered? • Pathway • Site of intracellular degradation • Functions • Involve in the regulation of the cell cycle
  3. 3. • Ubiquitin was first isolated from bovine thymus but later found in many different tissues and in many organisms. • Ubiquitin is a highly conserved protein • Ubiquitin is composed of 76 amino acid • Attachment site to target protein on ubiquitin is C-terminus • Bond is formed to side chain of Lys of target protein • Attachment is performed by array of enzymes (E1, E2, E3, E4).
  4. 4. • Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. • In general, this occurs by the hydrolysis of the peptide bond, and is most commonly achieved by cellular enzymes called proteases, but may also occur by intramolecular digestion, as well as by non-enzymatic methods such as the action of mineral acids and heat.
  5. 5. Avram Hershko Aaron Ciechanover Irwin Rose Nobel prize in chemistry, 2004
  6. 6. • A process where an enzyme system tags unwanted proteins with many molecules of the 76-amino acid residue protein ubiquitin. • The tagged proteins are then transported to the proteasome(a large multisubunit protease complex) where they are degraded into small peptides.
  7. 7. Unwanted protein
  8. 8. • E1: – ubiquitin-activating enzyme. – Activates the carboxy terminus of ubiquitin by reaction with ATP. – exists as two isoforms of 110- and 117-kDa, are found in both the nucleus and cytosol. – In mammals there is a single E1. • E2: – Ubiquitin-conjugating enzymes. – E2s are a super family of related proteins. There are eleven E2s in yeast, and 20-30 E2s in mammals.
  9. 9. • E3s: – Ubiquitin-protein ligases. – E3s play a key role in the ubiquitin pathway, as they are responsible for the selective recognition of protein substrates. • E4: – catalyzes the efficient polymerization of very long polyubiquitin chains, it has been characterized in yeast.
  10. 10. Unwanted protein
  11. 11. Three enzymes are involved, designated E1, E2 & E3.  Initially the terminal carboxyl group of ubiquitin is joined in a thioester bond to a cysteine residue on Ubiquitin-Activating Enzyme (E1). This is the ATP- dependent step.  The ubiquitin is then transferred to a sulfhydryl group on a Ubiquitin-Conjugating Enzyme (E2).
  12. 12. A Ubiquitin-Protein Ligase (E3) then promotes transfer of ubiquitin from E2 to the e-amino group of a Lys residue of a protein recognized by that E3, forming an isopeptide bond. There are many distinct Ubiquitin Ligases with differing substrate specificity. • One E3 is responsible for the N-end rule. • Some are specific for particular proteins. ubiquitin C S O Cys E2 H2N Lys protein to be degraded ubiquitin C O HS Cys E2N Lys protein to be degraded H + E3 + (Ubiquitin-Protein Ligase)
  13. 13. H2N COO destruction box chain of ubiquitins Primary structure of a protein targeted for degradation More ubiquitins are added to form a chain of ubiquitins. The terminal carboxyl of each ubiquitin is linked to the e-amino group of a lysine residue (Lys29 or Lys48) of the adjacent ubiquitin. A chain of 4 or more ubiquitins targets proteins for degradation in proteasomes.
  14. 14. H2N COO destruction box chain of ubiquitins Primary structure of a protein targeted for degradation Some proteins (e.g., mitotic cyclins involved in cell cycle regulation) have a destruction box sequence recognized by a domain of the corresponding Ubiquitin Ligase.
  15. 15.  Ubiquitin Ligases (E3) mostly consist of two families: 1) HECT (Homologous to the E6-AP Carboxyl Terminus)- domain containing a conserved Cys • Some Ubiquitin Ligases have a HECT domain containing a conserved Cys residue that participates in transfer of activated ubiquitin from E2 to a target protein.
  16. 16. 2) RING finger-domain Cys & His residues are ligands to two Zn++ ions stabilizes a molecular scaffold Some Ubiquitin Ligases contain a RING finger domain in which Cys & His residues are ligands to 2 Zn++ ions. A RING (Really Interesting New Gene) finger is not inherently catalytic. It stabilizes a characteristic globular domain conformation that serves as a molecular scaffold for residues that interact with E2.
  17. 17. • Ubiquitin—mediated degradation of cytosolic and membrane proteins occurs in the cytosol and on the cytosolic face of the ER membranes.
  18. 18. The 20S proteasome core complex encloses a cavity with 3 compartments joined by narrow passageways. Protease activities are associated with 3 of the b subunits, each having different substrate specificity. 20 S Proteasome (yeast) closed state two views PDB 1JD2   b  b  
  19. 19. 1. One catalytic b-subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide carbonyl) position.(hydrophobic a.a) 2. One has a trypsin-like activity with preference for arginine or lysine at the P1 position.(positive charged a. a) 3. One has a peptidoglutamyl peptidase like activity with preference for glutamate or other acidic residue at the P1 position. (negative charged a.a) Different variants of the 3 catalytic subunits, with different substrate specificity.
  20. 20. The poly-ubiquitinated protein is recognized by the 26S proteasome, unfolded and degraded. The ubiquitin molecules are recycled.
  21. 21. •Length of produced peptides: 3-23 amino acids •Average length of peptides: 7-9 amino acids •Peptide composition of given protein stays constant •Protein is completely degraded before import of next protein •Peptides produced by proteasome are further degraded by other proteases and amino peptidases. • Proteasome and immune system function: •Peptides of 8-9 amino acids in length are transported to the cell surface via the ER presented on the cell surface via MHC class I – molecules
  22. 22. • Eukaryotic cells also contain DUBs (Deubiquitinating enzymes), which are encoded by the UCH (Ubiquitin Carboxyl-terminal Hydrolases) and the UBP (UBiquitin-specific Processing proteases) gene families. • UCHs are relatively small proteins (< 40-kDa);in contrast, UBPs are 50-250-kDa proteins and constitute a large family. • Genome sequencing projects have identified more than 90 DUBs .
  23. 23. At the end of summer of 1979 in Fox Chase Cancer Center, Philadelphia. Seated left to right: Avram Hershko, Sandy Goldman, Jessie Warms, Hanna Heller, Standing left to right: Zelda Rose, Arthur Haas, Aaron Ciechanover, Mary Williamson, Irwin Rose, Keith Wilkinson and Leonard Cohen (last three people standing on the right side not identified)
  24. 24. • Advanced information on the Nobel Prize in Chemistry, 6 October 2004,kungl.vetenskapsahedemien,the royal swedish academy of science Information Department, P.O. Box 50005, SE-104 05 Stockholm, Sweden. • Biochemistry- Lubert Stryer • HARPER’S ILLUSTRATED BIOCHEMISTRY (28TH EDITION) by Robert murray,david A.bender,peter j kennekky,victor w rodwell,p.antony weil.

×